siRNA for inhibition of c-Met expression and anticancer composition containing the same

ABSTRACT

Disclosed are small interfering RNA (siRNA) that complementarily binds to a base sequence of c-Met transcript (mRNA transcript), thereby inhibiting expression of c-Met without inducing immune responses, and use of the siRNA for prevention and/or treatment of cancer. The siRNA that complementarily binds to c-Met-encoding mRNA may inhibit expression of c-Met, which is commonly overexpressed in almost all cancer cells, through RNA interference (RNAi), thereby inhibiting proliferation and metastasis of cancer cells, and thus, the siRNA may be useful as an anticancer agent.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a small interfering RNA (siRNA) thatcomplementary binds to a base sequence of c-Met transcript (mRNAtranscript), thereby inhibiting expression of c-Met without elicitingimmune responses, and use of the siRNA for prevention and/or treatmentof cancer.

(b) Description of the Related Art

c-Met is a proto-oncogene that encodes a protein known as hepatocytegrowth factor receptor (HGFR). Since it has been discovered for thefirst time in osteosarcoma of human treated with chemical carcinogen atthe year of 1984, it was found to be potential proto-oncogene due togenetic fusion with tpr (translocated promoter region) at the year of1986 (Cooper et al., Nature, 311, 29-33, 1984; Dean et al., Mol cellBiol., 7, 921-924, 1987; Park et al., Cell, 45, 895-904, 1986).

On binding to the cell surface receptor tyrosine kinase (TK) known asc-Met, hepatocyte growth factor (HGF) to be secreted by mesenchymalcells stimulates cell growth, cell motility, embryogenesis, woundhealing and angiogenesis. These pleiotropic actions are fundamentallyimportant during development, homeostasis, and tissue regeneration. HGFsignaling also contributes to oncogenesis and tumor progression inseveral human cancers and promotes aggressive cellular invasiveness thatis strongly linked to tumor metastasis. (Nakamura et al., J ClinInvest., 106, 1511-9, 2000; Comoglio et al., Semin Cancer Biol, 11,153-65, 2001; Boccaccio, Nat Rev Cancer, 6, 637-45, 2006; Huh C F etal., Proc Natl Acad Sci USA, 101, 4477-82, 2004).

Most of abnormal signal transduction of HGF/c-Met results from increasein the activity of HGF or c-Met due to overexpression or mutationthereof, and it is known to be closely related to a bad prognosis invarious cancer patients.

Owing to the discovery of close relationship between the activity ofc-Met protein and cancer incidence/metastasis and clinical success ofother receptor tyrosine kinase inhibitors, development of anticancerdrug targeting c-Met is actively progressed. However, currently, most ofdrug candidates in the clinical step are chemical synthesis inhibitors,such as tyrosine kinase inhibitor that inhibits c-Met signal pathway, tobe designed based on the tertiary structure of proteins or antibody toc-Met receptor.

Although low molecular kinase inhibitor has improved selectivity toc-Met compared to kinase inhibitors targeting a large panel of proteinkinases, there is still a concern for side effect due to off-targetingother protein which is structurally similar with c-Met protein.

Although antibody drugs comparatively have excellent selectivity, thereare problems in terms of productivity by the complicated productionprocess and instability during storage. Therefore, development ofeffective inhibitor targeting c-Met function is continuously demanded.

Recently, it has been revealed that the ribonucleic acid-mediatedinterference (RNAi) contributes to development of drug lead-candidate byexhibiting sequence specific gene silencing even for otherwisenon-druggable targets with the existing technologies. Therefore, RNAihas been considered as a technology capable of suggesting solutions tothe problems of limited targets and non-specificity in synthetic drugs,and overcoming limitations of chemical synthetic drugs, and thus, a lotof studies on the use thereof in development of medicines for variousdiseases that is hard to be treated by the existing technologies, inparticular cancer, are actively progressed.

However, it was found out that siRNA (small interfering RNA) triggersinnate immune responses, and also induces non-specific RNAi effect morefrequently than expected.

It has been reported that in mammal cells, long double stranded siRNAmay induce a deleterious interferon response; short double strandedsiRNA may also induce an initial interferon response deleterious to thehuman body or cells; and many siRNAs have been known to induce highernon-specific RNAi effect than expected (Kleirman et al. Nature,452:591-7, 2008).

Although there has been an attempt to develop siRNA anticancer drugstargeting c-Met which plays an important role in the progression ofcancer, so far the outcome is insignificant. Gene inhibition effect ofindividual sequence of siRNA has not been suggested, and particularly,immune activity has not been considered.

Although siRNA shows great promise as a novel medicine due to theadvantages such as high activity, excellent target specificity, and thelike, it has several obstacles to overcome for therapeutic development,such as low blood stability because it may be degraded by nuclease inblood, a poor ability to pass through cell membrane due to negativecharge, short half life in blood due to rapid excretion, whereby itslimited tissue distribution, and induction of off-target effect capableof affecting on regulation pathway of other genes.

Recently, in order to improve these disadvantages and enable theapplication to clinical test, studies are progressed on introducingchemical modification in siRNA (Davidson, Nat. Biotechnol., 24:951-952,2006; Sioud and Furset, J. Biomed. Biotechnol., 2006:23429, 2006).

SUMMARY OF THE INVENTION

Accordingly, the inventors developed siRNA that has high sequencespecificity and thus specifically binds to transcript of a target geneto increase RNAi activity, and does not induce any immune toxicity, andcompleted the invention.

One embodiment provides siRNA that complementarily binds to c-Met mRNAtranscript, thereby specifically inhibiting synthesis and/or expressionof c-Met.

Another embodiment provides an expression vector for expressing thesiRNA.

Another embodiment provides a pharmaceutical composition for inhibitingsynthesis and/or expression of c-Met, comprising the siRNA or the siRNAexpression vector as an active ingredient.

Another embodiment provides an anticancer composition comprising thesiRNA or the siRNA expression vector as an active ingredient.

Another embodiment provides a method for inhibiting synthesis and/orexpression of c-Met comprising preparing the above siRNA or the siRNAexpression vector; and contacting the siRNA or the siRNA expressionvector with c-Met-expressing cells, and use of the siRNA or the siRNAexpression vector for inhibition of synthesis and/or expression of c-Metin c-Met-expressing cells.

Yet another embodiment provides a method for inhibiting growth of cancercells comprising preparing the above siRNA or the siRNA expressionvector; and contacting the siRNA or the siRNA expression vector withc-Met-expressing cancer cells, and use of the siRNA or the siRNAexpression vector for inhibiting growth of cancer cells inc-Met-expressing cancer cells.

Yet another embodiment provides a method for preventing and/or treatingcancer comprising preparing the siRNA or the siRNA expression vector;and administering the siRNA or the siRNA expression vector to a patientin a therapeutically effective amount, and use of the siRNA or the siRNAexpression vector for prevention and/or treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a to FIG. 1 d shows change in cytokine concentration according tosiRNA treatment, wherein 1 a denotes the concentration of interferonalpha, 1 b denotes the concentration of interferon gamma, 1 c denotesthe concentration of interleukin-12, and 1 d denotes the concentrationof tumor necrosis factor.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides siRNA that complementarily binds to c-MetmRNA transcript base sequence, thereby inhibiting synthesis and/orexpression of c-Met in the cells, a pharmaceutical compositioncomprising the same, and use thereof.

According to one aspect of the present invention, provided is siRNA forspecifically inhibiting synthesis and/or expression of c-Met. Accordingto another aspect, provided is a pharmaceutical composition forinhibiting synthesis and/or expression of c-Met, comprising the siRNAspecifically inhibiting synthesis and/or expression of c-Met as anactive ingredient. According to yet another aspect, provided is an agentfor inhibiting cancer cell growth, or a pharmaceutical composition(anticancer composition) for prevention and/or treatment of a cancer,comprising the siRNA specifically inhibiting synthesis and/or expressionof c-Met as an active ingredient.

The present invention relates to a technology of inhibiting expressionof c-Met mRNA in mammals including human, an alternative splice form ora mutant thereof, or c-Met gene of the same lineage, which may beachieved by administering a specific amount of the siRNA of the presentinvention to a patient, to reduce the target mRNA.

Hereinafter, the present invention will be described in detail.

The c-Met may be originated from mammals, preferably human or it may bec-Met of the same lineage as human and a mutant thereof. The term ‘samelineage as human’ refers to mammals having genes or mRNA of 80% or moresequence homology with human c-Met genes or mRNA originated therefrom,and specifically, it may include human, primates, rodents, and the like.

According to one embodiment, cDNA sequence of a sense strandcorresponding to c-Met-encoding mRNA may be SEQ ID NO 1.

The siRNA according to the present invention may target mRNA or cDNAregion corresponding to at least one base sequence selected from thegroup consisting of a region consisting of consecutive 15 to 25 bp,preferably consecutive 18 to 22 bp, preferably consecutive 2 to 21 bp inthe mRNA or cDNA of c-Met. Preferable target regions on cDNA aresummarized in the following Table 1. Thus, according to one embodimentof the invention, provided is siRNA for targeting the mRNA or cDNAregion corresponding to at least one base sequence selected from thegroup consisting of SEQ ID NOs 2 to 21 in the c-Met cDNA region of SEQID NO: 1. Specifically, provided is siRNA for targeting the mRNA regioncorresponding to base sequence selected from the group consisting of SEQID NOs: 3, 18, and 21.

TABLE 1 Target regions on c-Met cDNA (SEQ ID NO: 1)(20) SEQ Start IDSequence nucleotide NO. (5′ -> 3′) in c-Met gene  2 GTAAAGAGGCACTAGCAAA  77  3 GCACTAGCAAAGTCCGAGA   86  4 CAGCAAAGCCAATTTATCA  306  5CTATGATGATCAACTCATT  375  6 CAATCATACTGCTGACATA  444  7CTCTAGATGCTCAGACTTT  803  8 TCTGGATTGCATTCCTACA  856  9CTGGATTGCATTCCTACAT  857 10 GCACAAAGCAAGCCAGATT 1039 11CTGCTTTAATAGGACACTT 1188 12 CAGGTTGTGGTTTCTCGAT 1390 13CTGGTTATCACTGGGAAGA 1507 14 TTGGTCCTGCCATGAATAA 1877 15AGACAAGCATCTTCAGTTA 2195 16 TCGCTCTAATTCAGAGATA 2376 17TCAGAGATAATCTGTTGTA 2386 18 GTGAGAATATACACTTACA 2645 19GGTGTTGTCTCAATATCAA 2809 20 CATTTGGATAGGCTTGTAA 2935 21CCAAAGGCATGAAATATCT 3566

As used herein, the term ‘target mRNA’ refers to human c-Met mRNA, c-MetmRNA of the same lineage as human, a mutant, or an alternative spliceform thereof. Specifically, it may include mutants of amino acid or basesequence such as NM_(—)000245, Mus musculus: NM_(—)008591, Macacamulatta: NM_(—)001168629, NM_(—)001127500: a splice form wherein basesequence 2262˜2317 are deleted, Y1230C/A3689G, D1228H/G3682C,V10921/G3274A, M1268T/T3795C, and the like. Thus, the siRNA of thepresent invention may target c-Met mRNA of human or the same lineage ashuman, an alternative splice form, or a mutant thereof.

As used herein, the wording ‘targeting mRNA (or cDNA) region’ means thatsiRNA has a base sequence complementary to the base sequence of thewhole or a part of the mRNA (or cDNA) region, for example, complementaryto 85˜100% of the whole base sequence, thus capable of specificallybinding to the mRNA (or cDNA) region.

As used herein, the term ‘complementary’ or ‘complementarily’ means thatboth strands of polynucleotide may form a base pair. Both strands ofcomplementary polynucleotide forms a Watson-Crick base pair to formdouble strands. When the base U is referred to herein, it may besubstituted by the base T unless otherwise indicated.

Since the inhibition effect on c-Met synthesis and/or expression andcancer therapeutic effect of the pharmaceutical composition of thepresent invention is achieved by effective inhibition on c-Met synthesisand/or expression, siRNA contained in the pharmaceutical composition asan active ingredient may be double stranded siRNA of 15-30 bp thattargets at least one of the specific mRNA regions as described above.According to a preferable embodiment, the siRNA may include at least oneselected from the group consisting of SEQ ID NOs 22 to 98. Morespecifically, the siRNA may be at least one selected from the groupconsisting of siRNA 1 to siRNA 40 as described in the following Table 2.

TABLE 2 SEQ Chemical ID siRNA structural NO Sequence (5′ -> 3′) Stranddesignation modification Double 22 GUAAAGAGGCACUAGCAAAdTdT Sense siRNA 1stranded 23 UUUGCUAGUGCCUCUUUACdTdT Antisense symmetric 24GCACUAGCAAAGUCCGAGAdTdT Sense siRNA 2 siRNA 25 UCUCGGACUUUGCUAGUGCdTdTAntisense (20) 26 CAGCAAAGCCAAUUUAUCAdTdT Sense siRNA 3 27UGAUAAAUUGGCUUUGCUGdTdT Antisense 28 CUAUGAUGAUCAACUCAUUdTdT SensesiRNA 4 29 AAUGAGUUGAUCAUCAUAGdTdT Antisense 30 CAAUCAUACUGCUGACAUAdTdTSense siRNA 5 31 UAUGUCAGCAGUAUGAUUGdTdT Antisense 32CUCUAGAUGCUCAGACUUUdTdT Sense siRNA 6 33 AAAGUCUGAGCAUCUAGAGdTdTAntisense 34 UCUGGAUUGCAUUCCUACAdTdT Sense siRNA 7 35UGUAGGAAUGCAAUCCAGAdTdT Antisense 36 CUGGAUUGCAUUCCUACAUdTdT SensesiRNA 8 37 AUGUAGGAAUGCAAUCCAGdTdT Antisense 38 GCACAAAGCAAGCCAGAUUdTdTSense siRNA 9 39 AAUCUGGCUUGCUUUGUGCdTdT Antisense 40CUGCUUUAAUAGGACACUUdTdT Sense siRNA 10 41 AAGUGUCCUAUUAAAGCAGdTdTAntisense 42 CAGGUUGUGGUUUCUCGAUdTdT Sense siRNA 11 43AUCGAGAAACCACAACCUGdTdT Antisense 44 CUGGUUAUCACUGGGAAGAdTdT SensesiRNA 12 45 UCUUCCCAGUGAUAACCAGdTdT Antisense 46 UUGGUCCUGCCAUGAAUAAdTdTSense siRNA 13 47 UUAUUCAUGGCAGGACCAAdTdT Antisense 48AGACAAGCAUCUUCAGUUAdTdT Sense siRNA 14 49 UAACUGAAGAUGCUUGUCUdTdTAntisense 50 UCGCUCUAAUUCAGAGAUAdTdT Sense siRNA 15 51UAUCUCUGAAUUAGAGCGAdTdT Antisense 52 UCAGAGAUAAUCUGUUGUAdTdT SensesiRNA 16 53 UACAACAGAUUAUCUCUGAdTdT Antisense 54 GUGAGAAUAUACACUUACAdTdTSense siRNA 17 55 UGUAAGUGUAUAUUCUCACdTdT Antisense 56GGUGUUGUCUCAAUAUCAAdTdT Sense siRNA 18 57 UUGAUAUUGAGACAACACCdTdTAntisense 58 CAUUUGGAUAGGCUUGUAAdTdT Sense siRNA 19 59UUACAAGCCUAUCCAAAUGdTdT Antisense 60 CCAAAGGCAUGAAAUAUCUdTdT SensesiRNA 20 61 AGAUAUUUCAUGCCUUUGGdTdT Antisense Double 62 CUAGCAAAGUCCGAGASense siRNA 21 stranded 25 UCUCGGACUUUGCUAGUGCdTdT Antisense asymmetric63 AGAAUAUACACUUACA Sense siRNA 22 siRNA 55 UGUAAGUGUAUAUUCUCACdTdTAntisense (3) 64 GAUUGCAUUCCUACAU Sense siRNA 23 61AGAUAUUUCAUGCCUUUGGdTdT Antisense Chemically 65 GCACUAGCAAAGUCCGAGAdT*dTSense siRNA24 siRNA2-mod1 modified 66 UCUCGGACUUUGCUAGUGCdT*dT AntisensesiRNA 67 GCACUAGCAAAGUCCGAGAdT*dT Sense siRNA25 siRNA2-mod2 (17) 68UCUCGGACUUUGCUAGUGCdT*dT Antisense 69 GCACUAGCAAAGUCCGAGAdT*dT SensesiRNA26 siRNA2-mod3 70 UCUCGGACUUUGCUAGUGCdT*dT Antisense 71GCACuAGCAAAGuCCGAGAdT*dT Sense siRNA27 siRNA2-mod4 72UCuCGGACuUUGCuAGuGCdT*dT Antisense 73 GCACUAGCAAAGUCCGAGAdT*dT SensesiRNA28 siRNA2-mod5 74 UCUCGGACUUUGCUAGUGCdT*dT Antisense 75GUGAGAAUAUACACUUACAdT*dT Sense siRNA29 siRNA17-mod1 76UGUAAGUGUAUAUUCUCACdT*dT Antisense 77 GUGAGAAUAUACACUUACAdT*dT SensesiRNA30 siRNA17-mod2 78 UGUAAGUGUAUAUUCUCACdT*dT Antisense 79GUGAGAAUAUACACUUACAdT*dT Sense siRNA31 siRNA17-mod3 80UGUAAGUGUAUAUUCUCACdT*dT Antisense 81 GuGAGAAuAuACACuuACAdT*dT SensesiRNA32 siRNA17-mod4 82 UGuAAGuGuAUAuuCuCACdT*dT Antisense 83GUGAGAAUAUACACUUACAdT*dT Sense siRNA33 siRNA17-mod5 84UGUAAGUGUAUAUUCUCACdT*dT Antisense 85 GUGAGAAUAUACACUUACAdT*dT SensesiRNA34 siRNA17-mod6 86 UGUAAGUGUAUAUUCUCACdT*dT Antisense 87CCAAAGGCAUGAAAUAUCUdT*dT Sense siRNA35 siRNA20-mod1 88AGAUAUUUCAUGCCUUUGGdT*dT Antisense 89 CCAAAGGCAUGAAAUAUCUdT*dT SensesiRNA36 siRNA20-mod2 90 AGAUAUUUCAUGCCUUUGGdT*dT Antisense 91CCAAAGGCAUGAAAUAUCUdT*dT Sense siRNA37 siRNA20-mod3 92AGAUAUUUCAUGCCUUUGGdT*dT Antisense 93 CCAAAGGCAuGAAAuAuCudT*dT SensesiRNA38 siRNA20-mod4 94 AGAuAuuuCAUGCCuuuGGdT*dT Antisense 95CCAAAGGCAUGAAAUAUCUdT*dT Sense siRNA39 siRNA20-mod5 96AGAUAUUUCAUGCCUUUGGdT*dT Antisense 97 CCAAAGGCAUGAAAUAUCUdT*dT SensesiRNA40 siRNA20-mod6 98 AGAUAUUUCAUGCCUUUGGdT*dT Antisense

The notation and contents of the chemical structural modification ofchemically modified siRNA (SEQ ID NOs 65 to 98) in the Table 2 aredescribed in the following Table 3 and Table 4.

TABLE 3 Notation Introduced chemical modification * Substitution of aphosphodiester linkage with a phosphorothioate linkage underlineSubstitution of 2′-OH of the ribose ring with 2′-O—Me Lower caseSubstitution of 2′-OH of the ribose ring with 2′-F letter Bold letterIntroduction of ENA(ethylene bridge nucleic acid)

TABLE 4 Structure name siRNA chemical modification mod1 2′-OH of riboseof 1^(st) and 2^(nd) nucleic acids of antisense strand are substitutedwith 2′-O—Me, and 3′ end dTdT (phosphodiester linkage) of sense andantisense strands are substituted with a phosphorothioate linkage(3′-dT*dT, *: phosphorothioate linkage) mod2 in addition to mod1modification, 2′-OH groups of riboses of 1st and 2nd nucleic acids ofsense strand are substituted with 2′-O—Me mod3 in addition to mod2modification, 2′-OH groups of riboses of all U containing nucleic acidsof sense strand are substituted with 2′-O—Me mod4 in addition to mod1modification, 2′-OH groups of riboses of all G containing nucleic acidsof sense and antisense strands are substituted with 2′-O—Me, and 2′-OHgroups of riboses of all U containing nucleic acids of sense andantisense strands are substituted with 2′-F. Provided that 10^(th),11^(th) bases of antisense strand are not substituted. mod5 in additionto mod2 modification, ENA(2′-O, 4′-C ethylene bridged nucleotide) isintroduced in one 5′ end nucleic acid of sense strand. mod6 2′-OH groupof ribose of 2^(nd) nucleic acid of antisense strand is substituted with2′-O—Me, and 3′ end dTdT (phosphodiester linkage) of sense and antisensestrands are substituted with a phosphorothioate linkage (3′-dT*dT, *:phosphorothioate linkage)

Since the siRNA has high sequence specificity for a specific targetregion of c-Met mRNA transcript, it can specifically complementarilybind to the transcript of a target gene, thereby increasing RNAinterference activity, thus having excellent activity of inhibitingc-Met expression and/or synthesis in cells. And, the siRNA has minimalimmune response inducing activity.

As described above, the siRNA of the present invention may be siRNAtargeting at least one region of mRNA selected from the group consistingof SEQ ID NOs. 2 to 21 of the c-Met cDNA region of SEQ ID NO. 1.Preferably, the siRNA may comprise at least one nucleotide sequenceselected from the group consisting of SEQ ID NOs. 22 to 98, and morepreferably, at least one selected from the group consisting of 40 kindsof siRNAs of SEQ ID NOs. 22 to 98. The siRNA includes ribonucleic acidsequence itself, and a recombinant vector (expression vector) expressingthe same. The expression vector may be a viral vector selected from thegroup consisting of a plasmid or an adeno-associated virus, aretrovirus, a vaccinia virus, an oncolytic adenovirus, and the like.

The pharmaceutical composition of the present invention may comprise thesiRNA as an active ingredient and a pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier may include any commonly usedcarriers, and for example, it may be at least one selected from thegroup consisting of water, a saline solution, phosphate buffered saline,dextrin, glycerol, ethanol, and the like, but not limited thereto.

The siRNA may be administered to mammals, preferably human, monkey, orrodents (mouse, rat), and particularly, to any mammals, for examplehuman, who has diseases or conditions related to c-Met expression, orrequires inhibition of c-Met expression.

To obtain c-Met inhibition effect while minimizing undesirable sideeffects such as an immune response, and the like, the concentration ofthe siRNA in the composition or the use or treatment concentration ofthe siRNA may be 0.001 to 1000 nM, preferably 0.01 to 100 nM, morepreferably 0.1 to 10 nM, but not limited thereto.

The siRNA or the pharmaceutical composition containing the same maytreat at least one cancer selected from the group consisting of varioussolid cancers such as lung cancer, liver cancer, colorectal cancer,pancreatic cancer, stomach cancer, breast cancer, ovarian cancer, renalcancer, thyroid cancer, esophageal cancer, prostate cancer, and thelike, osteosarcoma, soft tissue sarcoma, glioma, and the like.

Hereinafter, the structure and the designing process of the siRNA, and apharmaceutical composition containing the same will be described indetail.

The siRNA does not induce or do decrease the expression of c-Met proteinby degrading c-Met mRNA by RNAi pathway.

According to one embodiment, siRNA refers to small inhibitory RNAduplexes that induce RNA interference (RNAi) pathway. Specifically,siRNA is RNA duplexes comprising a sense strand and an antisense strandcomplementary thereto, wherein both strands comprise 15-30 bp,specifically 19-25 bp or 27 bp, more specifically 19-21 bp. The siRNAmay comprise a double stranded region and have a structure where asingle strand forms a hairpin or a stem-loop structure, or it may beduplexes of two separated strands. The sense strand may have identicalsequence to the nucleotide sequence of a target gene mRNA sequence. Aduplex forms between the sense strand and the antisense strandcomplementary thereto by Watson-Crick base pairing. The antisense strandof siRNA is captured in RISC(RNA-Induced Silencing Complex), and theRISC identifies the target mRNA which is complementary to the antisensestrand, and then, induces cleavage or translational inhibition of thetarget mRNA.

According to one embodiment, the double stranded siRNA may have anoverhang of 1 to 5 nucleotides at 3′ end, 5′ end, or both ends.Alternatively, it may have a blunt end truncated at both ends.Specifically, it may be siRNA described in US20020086356, and U.S. Pat.No. 7,056,704, which are incorporated herein by reference.

According to one embodiment, the siRNA comprises a sense strand and anantisense strand, wherein the sense strand and the antisense strand forma duplex of 15-30 bp, and the duplex may have a symmetrical structurehaving a blunt end without an overhang, or an asymmetric structurehaving an overhang of at least one nucleotide, for example 1-5nucleotides. The nucleotides of the overhang may be any sequence, but itmay have 2 dTs (deoxythymidine) attached thereto.

The antisense strand is hybridized with the target region of mRNA of SEQID NO. 1 under a physiological condition. The description ‘hybridizedunder physiological condition’ means that the antisense strand of thesiRNA is in vivo hybridized with a specific target region of mRNA.Specifically, the antisense strand may have 85% or more sequencecomplementarity to the target mRNA region, where the target mRNA regionis preferably at least one base sequence selected from SEQ ID NOs. 2 to21 as shown in Table 1, and more specifically, the antisense strand maycomprise a sequence completely complementary to consecutive 15 to 25 bp,preferably consecutive 18 to 22 bp within the base sequence of SEQ IDNO. 1. Still more preferably, the antisense strand of the siRNA maycomprise a sequence completely complementary to at least one basesequence selected from SEQ ID NOs. 2 to 21, as shown in Table 1.

According to one embodiment, the siRNA may have an asymmetric doublestranded structure, wherein one strand is shorter than the other strand.Specifically, in the case of siRNA (small interfering RNA) molecule ofdouble strands consisting of an antisense strand of 19 to 21 nucleotides(nt) and a sense strand of 15 to 19 nt having complementary sequence tothe antisense, the siRNA may be an asymmetric siRNA having a blunt endat 5′ end of the antisense and a 1-5 nucleotides overhang at 3′ end ofthe antisense. Specifically, it may be siRNA disclosed in WO09/078,685.

In the treatment using siRNA, it is required to select an optimum basesequence having highest activity in the base sequence of the targetedgene. Specifically, according to one embodiment, to increaserelationship between pre-clinical trials and clinical trial, it ispreferable to design c-Met siRNA comprising a conserved sequence betweenspecies. And, according to one embodiment, it is preferable to designsuch that the antisense strand binding to RISC may have high bindingability to RISC. Thus, it may be designed such that there may bedifference between thermodynamic stabilities between a sense strand andan antisense strand, thus increasing RISC binding ability of theantisense strand that is a guide strand, while the sense strand does notbind to RISC. Specifically, GC content of the sense strand may notexceed 60%; 3 or more adenine/guanine bases may exist in the 15^(th) to19^(th) positions from 5′ end of the sense strand; and G/C bases may beabundant in the 1^(st) to 7^(th) positions from 5′ end of the sensestrand. And, since due to repeated base sequences, internal sequences ofsiRNA itself may bind to each other and lower the ability ofcomplementary binding to mRNA, it may be preferable to design such thatless than 4 repeated base sequences exist. And, in the case of a sensestrand consisting of 19 bases, to bind to mRNA of a target gene toproperly induce degradation of the transcript, 3^(rd), 10^(th), and19^(th) bases from 5′ end of the sense strand may be adenine.

Further, according to one embodiment, siRNA has minimized non-specificbinding and immune response-inducing activity. The inducing of an immuneresponse of interferon, and the like by siRNA mostly occurs through TLR7(Toll-like receptor-7) that exists at endosome of antigen-presentingimmune cells, and binding of siRNA to TLR7 occurs in a sequence specificmanner like in GU rich sequences, and thus, it may be best to comprise asequence that is not recognized by TLR7. Specifically, it may not havean immune response-inducing sequence such as 5′-GUCCUUCAA-3′ and5′-UGUGU-3′, and have 70% or less complementarity to genes other thanc-Met.

Examples of the c-Met cDNA target sequence include the nucleotides ofthe sequences described in the above Table 1. Based on the targetsequences of Table 1, siRNA sequence may be designed such that siRNAlength may be longer or shorter than the length of the target sequence,or nucleotides complementary to the DNA sequences may be added ordeleted.

According to one embodiment of the invention, siRNA may comprise a sensestrand and an antisense strand, wherein the sense strand and theantisense strand form double strands of 15-30 bp without an overhang, orat least one end may have an overhang of 1-5 nucleotides, and theantisense strand may be hybridized to the mRNA region corresponding toany one of SEQ ID NOs 2 to 21, preferably SEQ ID NO 3, 18, 21, underphysiological condition. Namely, the antisense strand comprises asequence complementary to any one of SEQ ID NOs 2 to 21, preferably toSEQ ID NOs 3, 18, 21. Thus, the c-Met siRNA and the pharmaceuticalcomposition containing the same of the present invention do not induce aharmful interferon response and yet inhibit expression of c-Met gene.

The present invention inhibits expression of c-Met in cells bycomplementary binding to the mRNA region corresponding to at least onesequence selected from the group consisting of SEQ ID NO 3(5′-GCACTAGCAAAGTCCGAGA-3′), SEQ ID NO 18 (5′-GTGAGAATATACACTTACA-3′),and SEQ ID NO 21 (5′-CCAAAGGCATGAAATATCT-3′).

The c-Met siRNA according to specific embodiments of the invention areas described in the above Table 2.

According to one embodiment, the c-Met siRNA may be at least oneselected from the group consisting of siRNA 2 comprising a sensesequence of SEQ ID NO 24 and an antisense sequence of SEQ ID NO 25,siRNA 17 comprising a sense sequence of SEQ ID NO 54 and an antisensesequence of SEQ ID NO 55, siRNA 20 comprising a sense sequence of SEQ IDNO 60 and an antisense sequence of SEQ ID NO 61, siRNA 21 comprising asense sequence of SEQ ID NO 62 and an antisense sequence of SEQ ID NO25, siRNA 22 comprising a sense sequence of SEQ ID NO 63 and anantisense sequence of SEQ ID NO 55, and siRNA 23 comprising a sensesequence of SEQ ID NO 64 and an antisense sequence of SEQ ID NO 61.

Knockdown (c-Met expression inhibition) may be confirmed by measuringchange in the mRNA or protein level by quantitative PCR (qPCR)amplification, bDNA (branched DNA) assay, Western blot, ELISA, and thelike. According to one embodiment, a liposome complex is prepared totreat cancer cell lines, and then, ribonucleic acid-mediatedinterference of expression may be confirmed by bDNA assay in mRNA stage.

The siRNA sequence of the present invention has low immune responseinducing activity while effectively inhibiting synthesis or expressionof c-Met.

According to one embodiment, immune toxicity may be confirmed bytreating human peripheral blood mononuclear cells (PBMC) with ansiRNA-DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate) complex, and then, measuring whether released cytokinesof INF-α and INF-γ, tumor necrosis factor-α (TNF-α), interleukin-12(IL-12), and the like are increased or not in the culture fluid.

The siRNA may have a naturally occurring (unmodified) ribonucleic acidunit structure, or it may be chemically modified, and for example, itmay be synthesized such that the sugar or base structure of at least oneribonucleic acid, a linkage between ribonucleic acids may have at leastone chemical modification.

Through the chemical modification of siRNA, desirable effects such asimproved resistance to nuclease, increased intracellular uptake,increased cell targeting (target specificity), increased stability, ordecreased off-target effect such as decreased interferon activity,immune response and sense effect, and the like may be obtained withoutinfluencing the original RNAi activity.

The chemical modification method of siRNA is not specifically limited,and one of ordinary skills in the art may synthesize and modify thesiRNA as desired by a method known in the art (Andreas Henschel, FrankBuchholzl and Bianca Habermann (2004) DEQOR: a web based tool for thedesign and quality control of siRNAs. Nucleic Acids Research 32(WebServer Issue):W113-W120).

For example, a phosphodiester linkage of siRNA sense and antisensestrands may be substituted with a boranophosphate or a phosphorothioatelinkage to increase resistance to nucleic acid degradation. For example,a 3′ end phosphodiester linkage of siRNA sense and antisense strands maybe modified with a phosphorothioate linkage.

For another example, ENA (Ethylene bridge nucleic acid) or LNA (Lockednucleic acid) may be introduced at 5′ or 3′ end, or both ends of siRNAsense or antisense strand, and preferably, it may be introduced at 5′end of siRNA sense strand. Thereby, siRNA stability may be increased,and an immune response and non-specific inhibition may be reduced,without influencing the RNAi activity.

For yet another example, a 2′-OH group of ribose ring may be substitutedwith —NH₂ (amino group), —C-allyl(allyl group), —F (fluoro group), or—O-Me (or CH₃, methyl group). For example, 2′-OH group of ribose of 1stand 2nd nucleic acids of sense strand may be substituted with 2′-O-Me,2′-OH groups of ribose of 2^(nd) nucleic acid of antisense strand may besubstituted with 2′-O-Me, or 2′-OH of riboses of guanine (G) or uridine(U) containing nucleotides may be substituted with 2′-O-Me (methylgroup) or 2′-F (fluoro group).

In addition to the above described chemical modifications, variouschemical modifications may be made, and only one chemical modificationmay be made or a plurality of chemical modifications may be made incombination.

In the chemical modification, it is preferable that the activity ofknockdown of gene expression may not be reduced while stabilizing thedouble stranded structure of the siRNA, and thus, minimal modificationmay be preferred.

And, a ligand such as cholesterol, biotin, or cell penetrating peptidemay be attached to 5′- or 3′-end of sense strand of siRNA.

The siRNA of the present invention may be manufactured by in vitrotranscription or by cleaving long double stranded RNA with dicer orother nuclease having similar activities. Alternatively, as describedabove, siRNA may be expressed through a plasmid or a viral expressionvector, and the like.

A candidate siRNA sequence may be selected by experimentally confirmingwhether or not a specific siRNA sequence induces interferon in humanperipheral blood mononuclear cells (PBMC) comprising dendritic cells,and then, selecting sequences which do not induce an immune response.

Hereinafter, a drug delivery system (DDS) for delivering the siRNA willbe described.

A nucleic acid delivery system may be utilized to increase intracellulardelivery efficiency of siRNA.

The system for delivering nucleic acid into cells may include a viralvector, a non-viral vector, liposome, cationic polymer, micelle,emulsion, solid lipid nanoparticles, and the like. The non-viral vectormay have high delivery rate and long retention time. The viral vectormay include a retroviral vector, an adenoviral vector, a vaccinia virusvector, an adeno-associated viral vector, an oncolytic adenovirusvector, and the like. The nonviral vector may include plasmid. Inaddition, various forms such as liposome, cationic polymer, micelle,emulsion, solid lipid nanoparticles, and the like may be used. Thecationic polymer for delivering nucleic acid may include natural polymersuch as chitosan, atelocollagen, cationic polypeptide, and the like andsynthetic polymer such as poly(L-lysine), linear or branchedpolyethylene imine (PEI), cyclodextrin-based polycation, dendrimer, andthe like.

The siRNA or complex of the siRNA and nucleic acid delivery system(pharmaceutical composition) of the present invention may be in vivo orex vivo introduced into cells for cancer therapy. As shown by thefollowing Examples, if the siRNA or complex of the siRNA and nucleicacid delivery system of the present invention is introduced into cells,it may selectively decrease the expression of target protein c-Met ormodify mutation in the target gene to inhibit expression of c-Metinvolved in oncogenesis, and thus, cancer cells may be killed and cancermay be treated.

The siRNA or a pharmaceutical composition comprising the same of thepresent invention may be formulated for topical, oral or parenteraladministration, and the like. Specifically, the administration route ofsiRNA may be topical such as ocular, intravaginal, or intraanus, and thelike, parenteral such as intarpulmonary, intrabronchial, intranasal,intraepithelial, intraendothelial, intravenous, intraarterial,subcutaneous, intraabdominal, intramuscular, intracranial (intrathecalor intraventricular), and the like, or oral, and the like. For topicaladministration, the siRNA or the pharmaceutical composition comprisingthe same may be formulated in the form of a patch, ointment, lotion,cream, gel, drop, suppository, spray, solution, powder, and the like.For parenteral administration, intrathecal or intraventricularadministration, the siRNA or pharmaceutical composition containing thesame may comprise a sterilized aqueous solution containing appropriateadditives such as buffer, diluents, penetration enhancer, otherpharmaceutically acceptable carriers or excipients.

Further, the siRNA may be mixed with an injectable solution andadministered by intratumoral injection in the form of an injection, orit may be mixed with a gel or adhesive composition for transdermaldelivery and directly spread or adhered to an affected area to beadministered by transdermal route. The injectable solution is notspecifically limited, but preferably, it may be an isotonic aqueoussolution or suspension, and may be sterilized and/or contain additives(for example, antiseptic, stabilizer, wetting agent, emulsifying agent,solubilizing agent, a salt for controlling osmotic pressure, bufferand/or liposomalizing agent). The gel composition may contain aconventional gelling agent such as carboxymethyl cellulose, methylcellulose, acrylic acid polymer, carbopol, and the like and apharmaceutically acceptable carrier and/or a liposomalizing agent. And,in the adhesive composition for transdermal delivery, an activeingredient layer may include an adhesive layer, a layer for adsorbingsebum and a drug layer, and the drug layer may contain apharmaceutically acceptable carrier and/or a liposomalizing agent, butnot limited thereto.

Further, the siRNA or pharmaceutical composition comprising the same ofthe present invention may further comprise anticancer chemotherapeuticsin addition to the c-Met siRNA, or it may further comprise siRNA forinhibiting expression of at least one selected from the group consistingof growth factors, growth factor receptor, downstream signaltransduction protein, viral oncogene, and anticancer drug resistantgene.

Thus, combination of chemotherapy with c-Met siRNA may increasesensitivity to chemotherapeutics thus maximizing therapeutic effects anddecreasing side effects, and combination of siRNA for inhibitingexpression of various growth factors (VEGF, EGF, PDGF, and the like),growth factor receptor, downstream signal transduction protein, viraloncogene, and anticancer drug resistant gene with the siRNA forinhibiting the expression of c-Met of the present invention maysimultaneously block various cancer pathways to maximize anticancereffects.

The anticancer chemotherapeutics that may be used for combinedadministration with the siRNA for inhibiting the expression of c-Met ofthe present invention may include cisplatin, carboplatin, oxaliplatin,doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone,valubicin, curcumin, gefitinib, erlotinib, irinotecan, topotecan,vinblastine, vincristine, docetaxel, paclitaxel, and a combinationthereof.

According to another embodiment of the invention, provided is a methodfor inhibiting expression and/or synthesis of c-Met, comprisingpreparing the effective amount of the c-Met siRNA for inhibitingexpression and/or synthesis of c-Met; and contacting the siRNA withc-Met-expressing cells.

According to yet another embodiment, provided is a method for inhibitinggrowth of cancer cells, comprising preparing the effective amount of thec-Met siRNA for inhibiting synthesis and/or expression of c-Met; andcontacting the siRNA with c-Met-expressing cancer cells.

According to yet another embodiment, provided is a method for preventingand/or treating cancer, comprising preparing the c-Met siRNA; andadministering the siRNA to a patient in a therapeutically effectiveamount.

The method of preventing and/or treating cancer may further compriseidentifying a patient in need of prevention and/or treatment of cancerbefore the administration.

The cancer that may be treated according to the present invention may beat least one selected from the group consisting of most of the solidcancer (lung cancer, liver cancer, colorectal cancer, pancreatic cancer,stomach cancer, breast cancer, ovarian cancer, renal cancer, thyroidcancer, esophageal cancer, prostate cancer), osteosarcoma, soft tissuesarcoma, glioma, and the like.

The patient may include mammals, preferably, human, monkey, rodents(mouse, rat, and the like), and the like, and particularly, it mayinclude any mammals, for example, human having a disease or condition(for example, cancer) related to c-Met expression or requiringinhibition of c-Met expression.

The effective amount of the siRNA according to the present inventionrefers to the amount required for administration in order to obtain theeffect of inhibiting c-Met expression or synthesis or the resultingcancer cell growth inhibition and the effect of cancer therapy. Thus, itmay be appropriately controlled depending on various factors includingthe kind or severity of disease, kind of administered siRNA, kind ofdosage form, age, weight, general health state, gender and diet of apatient, administration time, administration route, and treatmentperiod, combined drug such as combined chemotherapeutic agents, and thelike. For example, daily dose may be 0.001 mg/kg 100 mg/kg, which may beadministered at a time or several times in divided dose.

The siRNA complementary to the base sequence of c-Met transcript (mRNA)of the preset invention may inhibit the expression of c-Met that iscommonly expressed in cancer cells by RNA-mediated interference (RNAi)to kill the cancer cells, and thus, it may exhibit excellent anticancereffect. And, it may minimize the induction of immune responses.

While most of the existing drugs inhibit the function of alreadyexpressed proteins, the RNAi technology of the present invention mayselectively inhibit the expression of specific disease inducing proteinswith high activity, and degrade the mRNA which is a pre-stage of proteinsynthesis, and thus, cancer growth and metastasis may be inhibitedwithout inducing side-effects, and it is expected to become a morefundamental cancer therapy.

Further, combination of chemotherapy with the c-Met siRNA may increasethe sensitivity to chemotherapeutics, to maximize therapeutic activityand reduce side-effects, and combination of siRNA for inhibiting theexpression of various growth factor (VEGF, EFG, PDGF, and the like),growth factor receptor and downstream signal transduction protein, viraloncogene, and anticancer agent resistant gene with the c-Met siRNA maysimultaneously block various cancer pathways, thus maximizing anticancereffect.

Hereinafter, the present invention will be described referring to thefollowing examples.

However, these examples are only to illustrate the invention, and thescope of the invention is not limited thereto.

Example 1 Design of Target Base Sequence to which siRNA for Inhibitingc-Met Expression May Bind

Using siRNA design programs of siDesign Center (Dharmacon), BLOCK-iT™RNAi Designer (Invitrogen), AsiDesigner (KRIBB), siDirect (University ofTokyo) and siRNA Target Finder (Ambion), a target base sequence to whichsiRNA may bind was derived from the c-Met mRNA sequence (NM_(—)000245).

TABLE 5 Target base sequence SEQ ID NO Sequence (5′ -> 3′)  2GTAAAGAGGCACTAGCAAA  3 GCACTAGCAAAGTCCGAGA  4 CAGCAAAGCCAATTTATCA  5CTATGATGATCAACTCATT  6 CAATCATACTGCTGACATA  7 CTCTAGATGCTCAGACTTT  8TCTGGATTGCATTCCTACA  9 CTGGATTGCATTCCTACAT 10 GCACAAAGCAAGCCAGATT 11CTGCTTTAATAGGACACTT 12 CAGGTTGTGGTTTCTCGAT 13 CTGGTTATCACTGGGAAGA 14TTGGTCCTGCCATGAATAA 15 AGACAAGCATCTTCAGTTA 16 TCGCTCTAATTCAGAGATA 17TCAGAGATAATCTGTTGTA 18 GTGAGAATATACACTTACA 19 GGTGTTGTCTCAATATCAA 20CATTTGGATAGGCTTGTAA 21 CCAAAGGCATGAAATATCT

Example 2 Manufacture of siRNA for Inhibiting c-Met Expression

23 kinds of siRNAs that may bind to the target base sequences designedin Example 1 were obtained from ST Pharm Co. Ltd (Korea). The 23 kindsof siRNA are as described in Table 6, wherein 3′ ends of both strandscomprise dTdT.

TABLE 6 Base sequence of siRNA for inhibiting c-Met expression SEQ siRNAID desig- NO Sequence (5′ -> 3′) Strand nation 22GUAAAGAGGCACUAGCAAAdTdT Sense siRNA 1 23 UUUGCUAGUGCCUCUUUACdTdTAntisense 24 GCACUAGCAAAGUCCGAGAdTdT Sense siRNA 2 25UCUCGGACUUUGCUAGUGCdTdT Antisense 26 CAGCAAAGCCAAUUUAUCAdTdT SensesiRNA 3 27 UGAUAAAUUGGCUUUGCUGdTdT Antisense 28 CUAUGAUGAUCAACUCAUUdTdTSense siRNA 4 29 AAUGAGUUGAUCAUCAUAGdTdT Antisense 30CAAUCAUACUGCUGACAUAdTdT Sense siRNA 5 31 UAUGUCAGCAGUAUGAUUGdTdTAntisense 32 CUCUAGAUGCUCAGACUUUdTdT Sense siRNA 6 33AAAGUCUGAGCAUCUAGAGdTdT Antisense 34 UCUGGAUUGCAUUCCUACAdTdT SensesiRNA 7 35 UGUAGGAAUGCAAUCCAGAdTdT Antisense 36 CUGGAUUGCAUUCCUACAUdTdTSense siRNA 8 37 AUGUAGGAAUGCAAUCCAGdTdT Antisense 38GCACAAAGCAAGCCAGAUUdTdT Sense siRNA 9 39 AAUCUGGCUUGCUUUGUGCdTdTAntisense 40 CUGCUUUAAUAGGACACUUdTdT Sense siRNA 10 41AAGUGUCCUAUUAAAGCAGdTdT Antisense 42 CAGGUUGUGGUUUCUCGAUdTdT SensesiRNA 11 43 AUCGAGAAACCACAACCUGdTdT Antisense 44 CUGGUUAUCACUGGGAAGAdTdTSense siRNA 12 45 UCUUCCCAGUGAUAACCAGdTdT Antisense 46UUGGUCCUGCCAUGAAUAAdTdT Sense siRNA 13 47 UUAUUCAUGGCAGGACCAAdTdTAntisense 48 AGACAAGCAUCUUCAGUUAdTdT Sense siRNA 14 49UAACUGAAGAUGCUUGUCUdTdT Antisense 50 UCGCUCUAAUUCAGAGAUAdTdT SensesiRNA 15 51 UAUCUCUGAAUUAGAGCGAdTdT Antisense 52 UCAGAGAUAAUCUGUUGUAdTdTSense siRNA 16 53 UACAACAGAUUAUCUCUGAdTdT Antisense 54GUGAGAAUAUACACUUACAdTdT Sense siRNA 17 55 UGUAAGUGUAUAUUCUCACdTdTAntisense 56 GGUGUUGUCUCAAUAUCAAdTdT Sense siRNA 18 57UUGAUAUUGAGACAACACCdTdT Antisense 58 CAUUUGGAUAGGCUUGUAAdTdT SensesiRNA 19 59 UUACAAGCCUAUCCAAAUGdTdT Antisense 60 CCAAAGGCAUGAAAUAUCUdTdTSense siRNA 20 61 AGAUAUUUCAUGCCUUUGGdTdT Antisense 62 CUAGCAAAGUCCGAGASense siRNA 21 25 UCUCGGACUUUGCUAGUGCdTdT Antisense 63 AGAAUAUACACUUACASense siRNA 22 55 UGUAAGUGUAUAUUCUCACdTdT Antisense 64 GAUUGCAUUCCUACAUSense siRNA 23 61 AGAUAUUUCAUGCCUUUGGdTdT Antisense

Example 3 c-Met Expression Inhibition Test in Cancer Cell Line UsingsiRNA

Using each siRNA manufactured in Example 2, human lung cancer cell line(A549, ATCC) and human liver cancer cell line (SK-Hep-1, ATCC) weretransformed, and c-Met expression was measured in the transformed cancercell line.

Example 3-1 Culture of Cancer Cell Line

Human lung cancer cell line (A549) and human liver cancer cell line(SK-Hep-1) obtained from American Type Culture Collection (ATCC) werecultured at 37° C., and 5% (v/v) CO₂, using RPM culture medium(GIBCO/Invitrogen, USA) containing 10% (v/v) fetal bovine serum,penicillin (100 units/ml) and streptomycin (100 ug/ml).

Example 3-2 Preparation of a Liposomal Complex of siRNA for c-Metexpression inhibition

25 ul of Opti-MEM medium (Gibco) each containing 10 nM of siRNA of thesiRNAs 1 to 23 of Example 2 and Opti-MEM medium containing 0.4 ul oflipofectamine 2000 (Invitrogen) per well were mixed in the same volume,and reacted at room temperature for 20 minutes to prepare a liposomalcomplex of siRNA.

Example 3-3 Inhibition of c-Met mRNA Expression in Cancer Cell LineUsing c-Met Targeting siRNA

The lung cancer cell line and liver cancer cell line cultured in Example3-1 were respectively seeded in a 96 well-plate at 10⁴ cells per well.After 24 hours, the medium was removed, and Opti-MEM medium was added inan amount of 50 μl per well. 5 μl of the liposomal complex of siRNAprepared in Example 3-2 was added, and cultured in a cell incubatorwhile maintaining at 37° C. and 5% (v/v) CO₂ for 24 hours.

To calculate IC₅₀ value, which is a drug concentration for 50%inhibition of c-Met mRNA expression, lung cancer cell line (A549) wastreated with each siRNA of the 7 concentrations between 0.0064 nM to 100nM.

Example 3-4 Quantitative Analysis of c-Met mRNA Expression in LungCancer Cell

The expression rate of c-Met mRNA, whose expression was inhibited by thesiRNA liposome complex, was measured by bDNA analysis using Quantigene2.0 system (Panomics, Inc.).

After treating the human lung cancer cell line with the 10 nM siRNAliposome complex for 24 hours, mRNA was quantified. According tomanufacturer's protocol, 1041 of a lysis mixture (Panomics, Quantigene2.0 bDNA kit) was treated per well of 96-well plate to lyze the cells at50° C. for 1 hour. Probe specifically binding to c-Met mRNA (Panomics,Cat. # SA-10157) was purchased from Panomics, Inc., and mixed togetherwith 80 μl of the obtained cell sample in a 96 well plate. Reaction wasperformed at 55° C. for 16 to 20 hours so that mRNA could be immobilizedin the well and bind to the probe. Subsequently, 100 μl of theamplification reagent of the kit was introduced in each well, reacted at55° C. and washed, which process was performed in two stages. 100 μl ofthe third amplification reagent was introduced and reacted at 50° C. andthen, 100 μl of a luminescence inducing reagent was introduced, andafter 5 minutes, luminescence was measured by a microplate reader(Bio-Tek, Synergy-HT) and expressed as percentage of that (100%) of thecontrol which was treated with lipofectamine only. The percentageindicates c-Met mRNA expression rate of each siRNA-treated test grouprelative to that of the control.

As shown in Table 7, it was confirmed that among 20 kinds of siRNA, 16kinds of siRNAs inhibit c-Met expression by 40% or less, 1 kind of siRNAinhibits c-Met expression by 40% to 70%, and 3 kinds of siRNAs inhibitc-Met expression by 70% or more.

Table 7 Relative expression rate of c-Met mRNAin human lung cancer cell line (A549) treated with 10 nM siRNA SEQc-Met mRN ID siRNA Aexpression NO Sequence (5′ -> 3′) No. rate (%)  2GAAAGAGGCACTAGCAAA  1  66.7  3 GCACTAGCAAAGTCCGAGA  2  22.7  4CAGCAAAGCCAATTTATCA  3  69.2  5 CTATGATGATCAACTCATT  4  81.4  6CAATCATACTGCTGACATA  5  68.5  7 CTCTAGATGCTCAGACTTT  6  71.5  8TCTGGATTGCATTCCTACA  7  81.8  9 CTGGATTGCATTCCTACAT  8  99.8 10GCACAAAGCAAGCCAGATT  9  84.6 11 CTGCTTTAATAGGACACTT 10  68.0 12CAGGTTGTGGTTTCTCGAT 11  68.8 13 CTGGTTATCACTGGGAAGA 12  67.1 14TTGGTCCTGCCATGAATAA 13  71.7 15 AGACAAGCATCTTCAGTTA 14  55.5 16TCGCTCTAATTCAGAGATA 15  68.1 17 TCAGAGATAATCTGTTGTA 16  99.9 18GTGAGAATATACACTTACA 17  12.8 19 GGTGTTGTCTCAATATCAA 18  60.5 20CATTTGGATAGGCTTGTAA 19 115.4 21 CCAAAGGCATGAAATATCT 20  26.6

For the 3 kinds of siRNAs 2, 17 and 20 having excellent gene expressioninhibition effect in Table 7, the extent of decreasing c-Met mRNAexpression was examined in the range of 100 nM to 0.0064 nM of siRNAsusing A549 cell line to calculate IC₅₀, and the results are described inthe following Table 8. The IC₅₀ value was calculated using SofrMax prosoftware supported by Spectra Max 190 (ELISA equipment) model. Comparingthe IC₅₀ values of siRNAs 2, 17 and 20 with those of siRNAs 14 and 15,it can be seen that the siRNAs 2, 17 and 20 show about 5 to 100 timehigher inhibition than siRNAs 14 and 15.

TABLE 8 IC₅₀(nM) in A549 cell line siRNA corresponding A549 SEQ ID NOsiRNA No. mRNA SEQ ID NO. (IC50: nM) 24, 25 2 3 0.29 54, 55 17 18 0.8760, 61 20 21 0.75 48, 49 14 15 4.35 50, 51 15 16 29

Example 3-5 Quantitative Analysis of c-Met mRNA Expression in LiverCancer Cell

Liver cancer cell line SK-Hep-1 was respectively treated with each 4 nMof siRNAs 2, 17 and 20 of a symmetric structure and siRNAs 21, 22 and 23of an asymmetric structure with sense strand shorter than antisensestrand, which target SEQ ID NO. 3, 18, or 21, and c-Met mRNA inhibitioneffect was examined, and the results are described in the followingTable 9. The experimental method was the same as Examples 3-4.

TABLE 9 siRNA c-Met expression SEQ ID NO siRNA No. Structural featurerate(%) 24, 25 2 Symmetric 35.7 62, 25 21 Asymmetric 31.3 54, 55 17Symmetric 32.3 64, 55 22 Asymmetric 43.8 60, 61 20 Symmetric 40.8 66, 6123 Asymmetric 60.8

As shown in the Table 9, if SEQ ID NOs. 3, 18, and 21 are targeted,asymmetric siRNAs could effectively inhibit c-Met expression to asimilar degree to symmetric siRNA.

Example 4 Inhibition Test of Cell Proliferation by siRNA

Cell proliferation inhibition effects by siRNAs 2, 14 and 15 weredetermined. Human lung cancer cells A549 were seeded in a 96 well plateat the number of 2.5×10³ per well, and after 24 hours, 0.4 μl of siRNAliposome complex prepared by the method of Example 3-2 was added to eachwell as designated concentrations of siRNA according to the method ofExample 3-3. 24 hours after the addition, media was replaced with 200 μlof fresh cell culture medium, and maintained in a cell incubator under37° C., 5% CO₂ for 5 days. And then, it was fixed with TCA(Trichloroacetic acid) for 30 minutes and stained with SRB(Sulforhodamine B, Sigma) at room temperature for 30 minutes. The wellwas washed with 10% (v/v) acetic acid 4-5 times, naturally dried,allowed to stand for one day, and then, 2000 of 10 mM unbuffered trissolution (Sigma) was introduced, absorbance was measured at 540 nm witha microplate reader (Bio-Tek, Synergy-HT), and expressed as percentageof control (100%) which was treated with Lipofectamine only.

The percentage means cell proliferation rate of the tes group treatedwith siRNAs 2, 14 or 15 relative to that of the control group. IC₅₀value was obtained using the percentage value calculated according tothe concentration of siRNA treated, and the results are described in thefollowing Table 10. As shown in the Table 10, siRNA 2 exhibits 20˜50time lower IC₅₀ value than siRNAs 14 and 15, thus indicating that cellproliferation inhibition effect of siRNA 2 on cell proliferation is20˜50 time higher than that of siRNAs 14 and 15. Therefore, the siRNA 2of the present invention may decrease c-Met mRNA expression, anddirectly induce inhibition of cancer cell proliferation due to thedecrease in c-Met expression thus exhibiting extraordinarily excellentanticancer effect.

TABLE 10 Cell division inhibition effect of c- Met-targeting siRNA(A549: IC₅₀(nM)) SEQ ID NO siRNA No. IC50 (nM) 48, 49 14 116 50, 51 1550.8 24, 25 2 2.44

Example 5 Effect of siRNA on Immunoactive Cytokine Release

To evaluate whether or not the siRNA of the present invention has immunetoxicity, experiment was conducted according to the followingprocedures.

Example 5-1 Preparation of Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMCs) were separated fromblood supplied by healthy volunteer at the experiment day usingHistopaque 1077 reagent (Sigma, St Louis, Mo., USA) by density gradientcentrifugation (Boyum A. Separation of leukocytes from blood and bonemarrow. Scand J Clin Lab Invest 21(Suppl 97):77, 1968). The blood wascarefully introduced on the Histopaque 1077 reagent transferred in a 15ml tube at 1:1 ratio (by weight) so as not to be mixed with each other.After centrifugation at room temperature, 400×g, for 30 minutes, onlythe PBMC containing layer was separated with a sterilized pipette. Intothe tube containing the separated PBMCs, 10 ml of phosphate bufferedsaline (PBS) was introduced, and then, the mixture was centrifuged at250×g for 10 minutes, and PBMCs were additionally washed twice with 5 mlof PBS. The separated PBMCs were suspended with serum-free x-vivo 15medium (Lonza, Walkersville, Md., USA) to a concentration of 4×10⁶cells/ml, and seeded in the volume of 100 ul per well in a 96-wellplate.

Example 5-2 Formulation of siRNA-DOTAP Complex

A complex of siRNA-DOTAP for transfecting PBMCs prepared in Example 5-1was prepared as follows. 5 ul of a DOTAP transfection reagent (ROCHE,Germany) and 45 ul of x-vivo 15 medium, and 1 ul (50 uM) of the siRNAand 49 ul of x-vivo 15 medium were respectively mixed, and then, reactedat room temperature for 10 minutes. After 10 minutes, the DOTAPcontaining solution and the siRNA containing solution were mixed andreacted at a temperature of 20 to 25° C. for 20 minutes to prepare asiRNA-DOTAP complex.

Example 5-3 Cell Culture

To 100 ul of the seeded PBMC culture media, the siRNA-DOTAP complexes ofthe siRNAs 2, 14 and 15 prepared according to Example 5-2 wererespectively added in the volume of 100 ul per well (the finalconcentration of siRNA was 250 nM), and then, cultured in a CO₂incubator of 37° C. for 18 hours. As control, cell culture groups nottreated with the siRNA-DOTAP complex and cell culture groups treatedwith DOTAP only without siRNA were used. And, Poly I:C(Polyinosinic-polycytidylic acid postassium salt, Sigma, USA) and APOB-1siRNA (sense GUC AUC ACA CUG AAU ACC AAU (SEQ ID NO 99), antisense: *AUUGGU AUU CAG UGU GAU GAC AC (SEQ ID NO 100), *: 5′ phosphates, providedby ST Pharm Co. Ltd.), known to induce an immune response, instead ofsiRNAwere formulated into a complex with DOTAP by the same method asExample 5-2, and cell culture groups were treated therewith and used aspositive control. After culture, only cell supernatant was separated.

Example 5-4 Measurement of Immune Activity

The amounts of interferon alpha (INF-α) and interferon gamma (INF-γ),tumor necrosis factor (TNF-α), and interleukin-12 (IL-12) released inthe supernatant were measured using Procarta Cytokine assay kit(Affymetrix, USA). Specifically, 50 ul of bead to which antibody tocytokine was attached (antibody bead) was transferred to a filter plateand washed with wash buffer once, and then, 50 ul of supernatant of thePMBC culture fluid and a cytokine standard solution were added andincubated at room temperature for 60 minutes while shaking at 500 rpm.

Then, the solution was washed with washing buffer once, 25 ul ofdetection antibody included in the kit was added, and reacted at roomtemperature for 30 minutes while shaking at 500 rpm. Again, the reactionsolution was removed under reduced pressure and washed, and then, 50 ulof streptavidin-PE (streptavidin phycoerythrin) included in the kit wasadded, and reacted at room temperature for 30 minutes while shaking at500 rpm, and then, the reaction solution was removed and washed threetimes. 120 ul of reading buffer was added and the reaction solution wasshaken at 500 rpm for 5 minutes, and then, PE fluorescence per cytokinebead was measured using Luminex equipment ((Bioplex luminex system,Biorad, USA), and the results are shown in FIGS. 1 a-1 d. The cytokineconcentration in the sample was calculated from a standard calibrationcurve of 1.22˜20,000 pg/ml range.

In FIGS. 1 a-1 d, ‘Medium’ denotes non-treated control, ‘DOTAP’ denotesonly DOTAP-treated group, ‘POLY I:C’ or ‘APOB-1’ denotes positivecontrol group, ‘siRNA 2’ denotes a test group treated with the siRNAs ofSEQ ID NOs. 24 and 25, ‘siRNA 14’ denotes a test group treated with thesiRNAs of SEQ ID NOs. 48 and 49, and ‘siRNA 15’ denotes a test grouptreated with the siRNA of SEQ ID NOs. 50 and 51. The FIGS. 1 a-1 d showscytokine level released in the PBMC, wherein 1 a denotes interferonalpha, 1 b denotes interferon gamma, 1 c denotes interleukin-12, and 1 ddenotes tumor necrosis factor.

The siRNA 2 exhibited very slight increase in all cytokines compared tocontrol and only DOTAP-only-treated group, and the increase is almostinsignificant compared to the increase of cytokine induced by POLY I:Cand APOB-1 used as positive control. And, comparing with siRNA 14 andsiRNA 15, it can be seen that increase in interferon alpha andinterferon gamma, particularly in interferon alpha, is remarkably low.Thus, it was confirmed that the siRNA 2 scarcely induces immune activityin human PBMC.

Example 6 Preparation of Chemically Modified siRNA for Inhibition ofc-Met Expression

The siRNAs 2, 17 and 20 prepared in Example 2 were designed so that thechemical structures may be modified in 6 forms (mod1˜6) as shown in theabove Table 4. The chemically modified siRNA was synthesized by ST PharmCo. Ltd (Korea). The 17 kinds of siRNAs chemically modified are shown inthe following Table 11, wherein the notation of the chemicalmodification is as explained in the above Table 3.

TABLE 11 SEQ ID NO Sequence (5′ -> 3′) siRNA designation 65GCACUAGCAAAGUCCGAGAdT*dT siRNA24 siRNA 2-mod1 66UCUCGGACUUUGCUAGUGCdT*dT 67 GCACUAGCAAAGUCCGAGAdT*dT siRNA25siRNA 2-mod2 68 UCUCGGACUUUGCUAGUGCdT*dT 69 GCACUAGCAAAGUCCGAGAdT*dTsiRNA26 siRNA 2-mod3 70 UCUCGGACUUUGCUAGUGCdT*dT 71GCACuAGCAAAGuCCGAGAdT*dT siRNA27 siRNA 2-mod4 72UCuCGGACuUUGCuAGuGCdT*dT 73 GCACUAGCAAAGUCCGAGAdT*dT siRNA28siRNA 2-mod5 74 UCUCGGACUUUGCUAGUGCdT*dT 75 GUGAGAAUAUACACUUACAdT*dTsiRNA29 siRNA 17-mod1 76 UGUAAGUGUAUAUUCUCACdT*dT 77GUGAGAAUAUACACUUACAdT*dT siRNA30 siRNA 17-mod2 78UGUAAGUGUAUAUUCUCACdT*dT 79 GUGAGAAUAUACACUUACAdT*dT siRNA31siRNA 17-mod3 80 UGUAAGUGUAUAUUCUCACdT*dT 81 GuGAGAAuAuACACuuACAdT*dTsiRNA32 siRNA 17-mod4 82 UGuAAGuGuAUAuuCuCACdT*dT 83GUGAGAAUAUACACUUACAdT*dT siRNA33 siRNA 17-mod5 84UGUAAGUGUAUAUUCUCACdT*dT 85 GUGAGAAUAUACACUUACAdT*dT siRNA34siRNA 17-mod6 86 UGUAAGUGUAUAUUCUCACdT*dT 87 CCAAAGGCAUGAAAUAUCUdT*dTsiRNA35 siRNA 20-mod1 88 AGAUAUUUCAUGCCUUUGGdT*dT 89CCAAAGGCAUGAAAUAUCUdT*dT siRNA36 siRNA 20-mod2 90AGAUAUUUCAUGCCUUUGGdT*dT 91 CCAAAGGCAUGAAAUAUCUdT*dT siRNA37siRNA 20-mod3 92 AGAUAUUUCAUGCCUUUGGdT*dT 93 CCAAAGGCAuGAAAuAuCudT*dTsiRNA38 siRNA 20-mod4 94 AGAuAuuuCAUGCCuuuGGdT*dT 95CCAAAGGCAUGAAAUAUCUdT*dT siRNA39 siRNA 20-mod5 96AGAUAUUUCAUGCCUUUGGdT*dT 97 CCAAAGGCAUGAAAUAUCUdT*dT siRNA40siRNA 20-mod6 98 AGAUAUUUCAUGCCUUUGGdT*dT

Example 7 Inhibition of C-Met mRNA Expression in Cancer Cell Line UsingChemically Modified siRNAs

To confirm whether or not the chemically modified siRNA retains mRNAinhibiting activity in cancer cell line, unmodified siRNA (siRNAs 2, 17and 20) of Example 2 and 17 siRNAs of siRNAs 24 to 40 chemicallymodified of Example 6 were respectively formulated into a liposomecomplex in the same manner as Example 3-2 to transfect human lung cancercell line (A549, ATCC) (10 nM siRNA), the c-Met expression in thetransfected cancer cell line was quantitatively analyzed in the samemanner as Example 3-4, and the results are described in the followingTable 12. In the Table 12, mod0 denotes chemically unmodified siRNA, andND denotes Not Detected.

TABLE 12 c-Met mRNA relative expression rate (%) in human lung cancercell line (A549) treated with 10 nM of chemically modified siRNA siRNA 2siRNA 17 siRNA 20 mod0 20.28 13.00 12.23 mod1 18.04 38.90 44.91 mod218.74 20.70 24.61 mod3 19.67 16.10 25.71 mod4 34.06 22.00 60.02 mod518.76 16.60 23.06 mod6 ND 15.50 20.00

As shown in the Table 12, even when siRNAs 2, 17 and 20 were chemicallymodified, the mRNA inhibition effects were retained in cancer cell line.Particularly, mod2, mod3, mod5, and mod6 exhibited effects equivalent toor better than the effect of unmodified siRNA.

Example 8 Inhibition Effect of Chemically Modified siRNA on ImmunoactiveCytokine Release

To investigate the degree of decrease in immune toxicity of siRNA due tochemical modification, siRNAs 2, 17 and 20 were respectivelystructurally modified to mod1-mod6, and then, human peripheral bloodmononuclear cells (PBMCs) were treated therewith to quantify releasedcytokine. The experiment was conducted in the same manner as Example 5,and the concentrations of cytokine (interferon alpha, interferon gamma,interleukin-12, tumor necrosis factor) released from PBMCs in theculture fluid were quantified and shown in the following Table 13. Inthe Table 13, ‘Medium’ denotes non-treated control, ‘DOTAP’ denotes onlyDOTAP-treated group, ‘POLY I:C’ or ‘APOB-1’ denotes positive controlgroup, ‘siRNA 2’ denotes a test group wherein the siRNA 2 is chemicallymodified with mod0-5, and ‘siRNA 20’ denotes a test group wherein thesiRNA 20 is chemically modified with mod0-6. The mod0 denotes chemicallyunmodified siRNA, and mod1-6 are as explained in the Table 4.

TABLE 13 Concentration (pg/ml) of cytokine released in cell culturefluid when PBMCs were treated with 250 nM of chemically structurallymodified siRNA INF-alpha INF-gamma IL-12p 40 TNF-alpha MEDIUM <1.2 10.915 32.6 DOTAP 9.1 18.3 43.4 131.0 siApoB-1 690.7 — — — POLY I:C — 46.9398.3 2691.5 siRNA 2 mod0 6.0 6.3 45.3 96.8 mod1 11.1 6.3 65.8 128.2mod2 21.4 50.5 75.2 154.1 mod3 8.7 7.3 67.3 124.9 mod4 7.8 11.8 54.694.2 mod5 7.8 8.3 73.6 147.4 siRNA 17 mod0 1091.3 21.5 29.8 146.0 mod1413.2 16.3 30.0 136.9 mod2 23.9 11.8 88.8 181.0 mod3 4.0 10.1 78.0 140.1mod4 7.0 8.3 59.1 93.9 mod5 60.3 10.1 59.1 147.4 mod6 10.1 1.9 20.1 84.9siRNA 20 mod0 597.7 16.6 37.5 136.3 mod1 6.0 10.1 57.4 153.6 mod2 6.514.9 61.0 108.5 mod3 8.7 8.3 60.5 115.6 mod4 6.5 10.1 53.8 87.0 mod523.9 13.4 73.1 161.6 mod6 21.4 1.9 27.9 103.9

As shown in the Table 13, the siRNA 2 exhibited no change or very slightincrease in all cytokines, compared to the control and onlyDOTAP-only-treated group.

Meanwhile, the siRNAs 17 and 20 exhibited rapid decrease in interferonalpha due to the chemical modification. For the other cytokines, thereis no significant change or very slight increase. Thus, it was confirmedthat the chemical modification of the siRNAs 17 and 20 may remarkablydecrease immune activity.

Example 9 Inhibition of Off-Target Effect by Sense Strand of ChemicallyModified siRNA

The following experiment was conducted to examine whether or notoff-target effect by sense strand may be removed through chemicalmodification of siRNA.

Example 9-1 Preparation of Firefly Luciferase Vector

A sequence complementary to an antisense strand and a sequencecomplementary to a sense strand of siRNA were respectively cloned in apMIR-REPORT (Ambion) vector expressing firefly luciferase to prepare twodifferent plasmids. The complementary sequences were designed andsynthesized by Cosmo Genetech such that both ends had SpeI and HindIIIrestriction sites overhang, and then, cloned using SpeI and HindIIIrestriction sites of a pMIR-REPORT vector.

Example 9-2 Measurement of Inhibition of Off-Target Effect ThroughChemical Modification of siRNA

Using plasmids comprising respective sequences complementary to eachsense strand and antisense strand of siRNA, prepared in Example 9-1,effects of the antisense and sense strands of siRNA were measured. Thedegree of off-target effect by sense strand can be seen by confirmingthat if a sense strand binds to RISC and acts on a sequence having abase sequence complementary to the sense strand, the amount ofluciferase expressed by firefly Luciferase plasmid having a sequencecomplementary to the sense strand decreases compared to the cell that isnot treated with the siRNA. And, for cells treated with fireflyluciferase plasmid having a sequence complementary to antisense, thedegree of retention of siRNA activity by antisense after chemicalmodification may be confirmed by degree of reduction in luciferaseexhibited by the siRNA.

Specifically, the firefly luciferase vector prepared in Example 9-1 wastransfected in HeLa and A549 cells (ATCC) together with the siRNA, andthen, the amount of expressed firefly luciferase was measured byluciferase assay. One day before transfection, the HeLa and A549 celllines were prepared in a 24 well plate at 6*10⁴ cells/well. Theluciferase vector (100 ng) in which complementary base sequences werecloned were transfected in Opti-MEM medium (Gibco) using lipofectamine2000 (Invitrogen) together with a vector for normalization, pRL-SV40vector (2 ng, Promega) expressing renilla luciferase. After 24 hours,the cells were lyzed using passive lysis buffer (Promega), and then,luciferase activity was measured by dual luciferase assay kit (Promega).

The measured firefly luciferase value was normalized for transfectionefficiency with the measured renilla luciferase value, and then,percentage value to the normalized luciferase value (100%) of control,which was transfected with renilla luciferase vector and fireflyluciferase vector in which sequences complementary to each strand werecloned without siRNA, was calculated and described in the followingTable 14. In the Table 14, mod0 denotes chemically unmodified siRNA, andmod1˜6 are as explained in the Table 4.

TABLE 14 Off-target effect decrease through chemical modification ofsiRNA Luciferase activity (%) HeLa A549 Name of Plasmid comprisingPlasmid comprising Plasmid comprising Plasmid comprising chemicallysequence sequence sequence sequence siRNA modified complementary tocomplementary to complementary complementary to No. structure sensestrand antisense strand to sense strand antisense strand 2 mod0 118.49.1 139.3 5.9 20 mod0 21.08 7.68 17.56 7.08 mod1 8.19 30.29 9.6 65.01mod2 48.38 12.45 80.91 26.14 mod3 31.34 19.03 38.23 15.81 mod4 12.2347.58 16.27 56.91 mod5 56.73 8.14 63.49 17.64

As shown in the Table 14, in human lung cancer cell line A549 anduterine cervical cancer cell line HeLa, unmodified siRNA (mod0) per sehad no off-target effect by sense strand in case of siRNA 2. However, inthe case of siRNA 20, slight off-target effect by sense strand was seenthrough decrease in the activity of firefly luciferase having sequencecomplementary to the sense strand, but if chemically modified, sensestrand effect was decreased and antisense effect was maintained,particularly in mod2 and 5.

1. A double stranded siRNA (small interfering RNA) of 15 to 30 bp, whichtargets an mRNA region corresponding to at least one selected from c-MetcDNA regions described in the following Table 1-1: TABLE 1-1Sequence No. Sequence (5′ -> 3′)  2 GAAAGAGGCACTAGCAAA  3GCACTAGCAAAGTCCGAGA  4 CAGCAAAGCCAATTTATCA  5 CTATGATGATCAACTCATT  6CAATCATACTGCTGACATA  7 CTCTAGATGCTCAGACTTT  8 TCTGGATTGCATTCCTACA  9CTGGATTGCATTCCTACAT 10 GCACAAAGCAAGCCAGATT 11 CTGCTTTAATAGGACACTT 12CAGGTTGTGGTTTCTCGAT 13 CTGGTTATCACTGGGAAGA 14 TTGGTCCTGCCATGAATAA 15AGACAAGCATCTTCAGTTA 16 TCGCTCTAATTCAGAGATA 17 TCAGAGATAATCTGTTGTA 18GTGAGAATATACACTTACA 19 GGTGTTGTCTCAATATCAA 20 CATTTGGATAGGCTTGTAA 21CCAAAGGCATGAAATATCT


2. The siRNA according to claim 1, wherein the siRNA targets an mRNAregion corresponding to at least one base sequence selected from thegroup consisting of SEQ ID NOs 3, 18 and
 21. 3. The siRNA according toclaim 1, wherein the siRNA comprises an overhang consisting of 1 to 5nucleotides at 3′ end, 5′ end, or both ends.
 4. The siRNA according toclaim 2, wherein the siRNA comprises nucleotide sequence selected fromthe group consisting of siRNAs 1 to 23 described in the following Table6: TABLE 6 Sequence siRNA No. Sequence (5′ ->3′) Strand designation 22GUAAAGAGGCACUAGCAAAdTdT Sense siRNA 1 23 UUUGCUAGUGCCUCUUUACdTdTAntisense 24 GCACUAGCAAAGUCCGAGAdTdT Sense siRNA 2 25UCUCGGACUUUGCUAGUGCdTdT Antisense 26 CAGCAAAGCCAAUUUAUCAdTdT SensesiRNA 3 27 UGAUAAAUUGGCUUUGCUGdTdT Antisense 28 CUAUGAUGAUCAACUCAUUdTdTSense siRNA 4 29 AAUGAGUUGAUCAUCAUAGdTdT Antisense 30CAAUCAUACUGCUGACAUAdTdT Sense siRNA 5 31 UAUGUCAGCAGUAUGAUUGdTdTAntisense 32 CUCUAGAUGCUCAGACUUUdTdT Sense siRNA 6 33AAAGUCUGAGCAUCUAGAGdTdT Antisense 34 UCUGGAUUGCAUUCCUACAdTdT SensesiRNA 7 35 UGUAGGAAUGCAAUCCAGAdTdT Antisense 36 CUGGAUUGCAUUCCUACAUdTdTSense siRNA 8 37 AUGUAGGAAUGCAAUCCAGdTdT Antisense 38GCACAAAGCAAGCCAGAUUdTdT Sense siRNA 9 39 AAUCUGGCUUGCUUUGUGCdTdTAntisense 40 CUGCUUUAAUAGGACACUUdTdT Sense siRNA 10 41AAGUGUCCUAUUAAAGCAGdTdT Antisense 42 CAGGUUGUGGUUUCUCGAUdTdT SensesiRNA 11 43 AUCGAGAAACCACAACCUGdTdT Antisense 44 CUGGUUAUCACUGGGAAGAdTdTSense siRNA 12 45 UCUUCCCAGUGAUAACCAGdTdT Antisense 46UUGGUCCUGCCAUGAAUAAdTdT Sense siRNA 13 47 UUAUUCAUGGCAGGACCAAdTdTAntisense 48 AGACAAGCAUCUUCAGUUAdTdT Sense siRNA 14 49UAACUGAAGAUGCUUGUCUdTdT Antisense 50 UCGCUCUAAUUCAGAGAUAdTdT SensesiRNA 15 51 UAUCUCUGAAUUAGAGCGAdTdT Antisense 52 UCAGAGAUAAUCUGUUGUAdTdTSense siRNA 16 53 UACAACAGAUUAUCUCUGAdTdT Antisense 54GUGAGAAUAUACACUUACAdTdT Sense siRNA 17 55 UGUAAGUGUAUAUUCUCACdTdTAntisense 56 GGUGUUGUCUCAAUAUCAAdTdT Sense siRNA 18 57UUGAUAUUGAGACAACACCdTdT Antisense 58 CAUUUGGAUAGGCUUGUAAdTdT SensesiRNA 19 59 UUACAAGCCUAUCCAAAUGdTdT Antisense 60 CCAAAGGCAUGAAAUAUCUdTdTSense siRNA 20 61 AGAUAUUUCAUGCCUUUGGdTdT Antisense 62 CUAGCAAAGUCCGAGASense siRNA 21 25 UCUCGGACUUUGCUAGUGCdTdT Antisense 63 AGAAUAUACACUUACASense siRNA 22 55 UGUAAGUGUAUAUUCUCACdTdT Antisense 64 GAUUGCAUUCCUACAUSense siRNA 23 61 AGAUAUUUCAUGCCUUUGGdTdT Antisense


5. The siRNA according to claim 4, wherein the siRNA is selected fromthe group consisting of siRNA 2 comprising a sense sequence of SEQ ID NO24 and an antisense sequence of SEQ ID NO 25; siRNA 17 comprising asense sequence of SEQ ID NO 54 and an antisense sequence of SEQ ID NO55; siRNA 20 comprising a sense sequence of SEQ ID NO 60 and anantisense sequence of SEQ ID NO 61; siRNA 21 comprising a sense sequenceof SEQ ID NO 62 and an antisense sequence of SEQ ID NO 25; siRNA 22comprising a sense sequence of SEQ ID NO 63 and an antisense sequence ofSEQ ID NO 55; and siRNA 23 comprising a sense sequence of SEQ ID NO 64and an antisense sequence of SEQ ID NO
 61. 6. The siRNA according toclaim 1, wherein the sugar or base structure of at least oneribonucleotide, or a linkage between the ribonucleotides is chemicallymodified.
 7. The siRNA according to claim 6, wherein the chemicalmodification is modification of a phosphodiester linkage at 3′ end, 5′end or both ends with a boranophosphate or a phosphorothioate linkage.8. The siRNA according to claim 6, wherein the chemical modification isintroduction of ENA (Ethylene bridge nucleic acid) at 3′ end, 5′ end, orboth ends.
 9. The siRNA according to claim 6, wherein the chemicalmodification is substitution of 2′-OH (hydroxyl group) of the ribosering with at least one selected from the group consisting of —NH₂ (aminogroup), —C-allyl group, —F (fluoro group), and —O-Me (methyl group). 10.The siRNA according to claim 6, wherein the chemically modified siRNAcomprises nucleotide sequence selected from the group consisting ofsiRNA 24 to 40 described in the following Table 11-1. TABLE 11-1 Se-siRNA quence desig- No. Sequence (5′ -> 3′) nation 65GCACUAGCAAAGUCCGAGAdT*dT siRNA24 66 UCUCGGACUUUGCUAGUGCdT*dT 67GCACUAGCAAAGUCCGAGAdT*dT siRNA25 68 UCUCGGACUUUGCUAGUGCdT*dT 69GCACUAGCAAAGUCCGAGAdT*dT siRNA26 70 UCUCGGACUUUGCUAGUGCdT*dT 71GCACuAGCAAAGuCCGAGAdT*dT siRNA27 72 UCuCGGACuUUGCuAGuGCdT*dT 73GCACUAGCAAAGUCCGAGAdT*dT siRNA28 74 UCUCGGACUUUGCUAGUGCdT*dT 75GUGAGAAUAUACACUUACAdT*dT siRNA29 76 UGUAAGUGUAUAUUCUCACdT*dT 77GUGAGAAUAUACACUUACAdT*dT siRNA30 78 UGUAAGUGUAUAUUCUCACdT*dT 79GUGAGAAUAUACACUUACAdT*dT siRNA31 80 UGUAAGUGUAUAUUCUCACdT*dT 81GuGAGAAuAuACACuuACAdT*dT siRNA32 82 UGuAAGuGuAUAuuCuCACdT*dT 83GUGAGAAUAUACACUUACAdT*dT siRNA33 84 UGUAAGUGUAUAUUCUCACdT*dT 85GUGAGAAUAUACACUUACAdT*dT siRNA34 86 UGUAAGUGUAUAUUCUCACdT*dT 87CCAAAGGCAUGAAAUAUCUdT*dT siRNA35 88 AGAUAUUUCAUGCCUUUGGdT*dT 89CCAAAGGCAUGAAAUAUCUdT*dT siRNA36 90 AGAUAUUUCAUGCCUUUGGdT*dT 91CCAAAGGCAUGAAAUAUCUdT*dT siRNA37 92 AGAUAUUUCAUGCCUUUGGdT*dT 93CCAAAGGCAuGAAAuAuCudT*dT siRNA38 94 AGAuAuuuCAUGCCuuuGGdT*dT 95CCAAAGGCAUGAAAUAUCUdT*dT siRNA39 96 AGAUAUUUCAUGCCUUUGGdT*dT 97CCAAAGGCAUGAAAUAUCUdT*dT siRNA40 98 AGAUAUUUCAUGCCUUUGGdT*dT

In the above Table 11-1, notation of chemical modification is asdescribed in the following Table 3: TABLE 3 notation Introduced chemicalmodification * Substitution of a phosphodiester linkage with aphosphorothioate linkage underline Substitution of 2′-OH of the ribosering with 2′-O—Me Lower case Substitution of 2′-OH of the ribose ringwith 2′-F letter Bold letter Introduction of ENA(ethylene bridge nucleicacid)


11. An expression vector comprising the siRNA according to claim
 1. 12.The expression vector according to claim 11, wherein the expressionvector is selected from the group consisting of a plasmid, anadeno-associated virus vector, a retrovirus vector, a vaccinia virusvector, and an oncolytic adenovirus vector.
 13. An anticancercomposition containing the siRNA according to claim 1 as an activeingredient.
 14. The anticancer composition according to claim 13,comprising the siRNA in the form of a complex with a nucleic aciddelivery system.
 15. The anticancer composition according to claim 14,wherein the nucleic acid delivery system is selected from the groupconsisting of a viral vector, a non-viral vector, liposome, cationicpolymer, micelle, emulsion, and solid lipid nanoparticles.
 16. Theanticancer composition according to claim 13, further comprisinganticancer chemotherapeutics, or siRNA for inhibiting the expression ofone selected from the group consisting of growth factor, growth factorreceptor, downstream signal transduction protein, viral oncogene, andanticancer agent resistant gene.
 17. A method for inhibiting synthesisand/or expression of c-Met, comprising preparing the siRNA according toclaim 1; and contacting the siRNA with c-Met-expressing cells.
 18. Amethod for inhibiting growth of cancer cells, comprising preparing thesiRNA according to claim 1; and contacting the siRNA withc-Met-expressing cancer cells.
 19. A method for preventing and/ortreating cancer, comprising preparing the siRNA according to claim 1;and administering the siRNA to a patient in a therapeutically effectiveamount.